Species-specif ic COl primers for rapid identif ication of a globally signif icant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

WANG Yu-sheng, TlAN Hu, WAN Fang-hao, ZHANG Gui-fen

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests/Key Laboratory of Integrated Pest Management of Crop, Ministry of Agriculture/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2 Caofeidian Sub-Center of Hebei Entry-Exit Inspection and Quarantine Technical Center, Tangshan 063200, P.R.China

Abstract The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava, and its recent introduction into Asia has raised considerable alarm. To slow or prevent further invasion, an accurate, simple, and developmental-stage-independent detection method for P. manihoti is required. In the present study, a PCR method based on a species-specif ic mitochondrial DNA cytochrome oxidase I (SS-COI) marker was developed for rapid identif ication of P. manihoti. One pair of SS-COI primers (PMSSZW-1F and PMSSZW-1R) was designed based on sequence variations in the COI gene among P. manihoti and related mealybug species. Specif icity of the primer pair was validated on 21 closely related species. Sensitivity tests were performed on four immature developmental stages and female adults. Eff icacy tests demonstrated that at the relatively low concentration of (135.2±14.7) pg μL-1 resuspended DNA, the specif ic fragment was detected in all replicates. Furthermore, the SS-COI primer pair was assayed on three populations of P. manihoti from major exporting countries of cassava. The PCR assay was proved to be a rapid, simple, and reliable molecular measure for the identif ication of P. manihoti. This tool will be useful for quarantine, monitoring, and management of this invasive pest.

Keywords: Phenacoccus manihoti, cassava mealybug, invasive pest, molecular identif ication, species-specif ic COI primers

1. lntroduction

The cassava mealybug, Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), which originated in South America (L?hr et al. 1990), has become one of the most important pests of cassava Manihot esculenta Crantz worldwide (Bellotti et al. 1999). P. manihoti is a polyphagous species, feeding on plants of nine families (Morales et al. 2016), but cassava is its preferred host (Cox and Williams 1981; Le Ru and Tertuliano 1993). P. manihoti poses a threat to the food security and livelihood of much of the global poor (Yonow et al. 2017). It is notorious for crop devastation in invaded regions of Africa (Nwanze 1982; Neuenschwander et al. 1988; Herren and Neuenschwander 1991; Nweke 2009) and Asia (Muniappan et al. 2009; Winotai et al. 2010; Bellotti et al. 2012; Parsa et al. 2012). It has become naturalized throughout Africa since it was accidentally introduced into Congo in the early 1970s (Matile-Ferrero 1978; Herren and Neuenschwander 1991). Usually, the yield reduction caused by this mealybug is about 80% (Nwanze 1982), leading to economic losses that can exceed two billion US dollars per year (Herren 1981; Neuenschwander et al. 1988; Herren and Neuenschwander 1991). In Asia, P. manihoti was f irst detected in Thailand in 2008 (Parsa et al. 2012) and soon spread aggressively into neighboring countries, such as Indonesia, Cambodia, Laos, and Vietnam (Muniappan et al. 2009; Parsa et al. 2012) and caused serious damage to cassava production (Winotai et al. 2010; Bellotti et al. 2012). However, this species has not yet invaded China.

To date, at least 34 countries or regions have been invaded by P. manihoti (Morales et al. 2016) and it could impose a serious threat to the cassava industry in yet uninvaded countries (Parsa et al. 2012). P. manihoti has thelytokous parthenogenesis, so introduction of a single individual could be suff icient to lead to successful invasion. Moreover, ovisacs or eggs adhere to carriers, while nymphs and adults can spread with wind, facilitating long-distance dispersal (Parsa et al. 2012). In Africa, P. manihoti is spreading at an alarming rate of 150 km per year (Winotai et al. 2010), compared to less than 30 km per year for other invasive Hemipterans (Liebhold and Tobin 2008). Risk analysis for biological invasion indicates that the regions predicted suitable for P. manihoti, including China, coincide with high risk areas for cassava (Wu and Wang 2011; Parsa et al. 2012; Zhou et al. 2014a, b).

A rapid and effective diagnostic method is urgently needed to forestall further spread. Morphological characteristics are commonly used for identif ication of this pest; however, this method is limited by high degrees of similarity among related mealybug species. Molecular identif ication methods are sensitive and simple enough to be performed routinely by a non-specialist (Rugman-Jones et al. 2006; Zhang et al. 2012; Jiang et al. 2013). Random amplif ied polymorphic DNA (RAPD) markers have been used to distinguish the cassava mealybug (Calatayud and Le Rü 2006). However, RAPD analysis is sensitive to changes in reaction conditions or even models of the PCR thermal cycler (Black 1993). Species-specif ic PCR (SS-PCR) with species-specif ic primers has emerged as a promising tool for identif ication. This rapid method identif ies species based solely on the presence of a specif ic band in gel electrophoresis with no need for additional digestion or sequencing (Hosseini et al. 2007). SS-PCR is a species-specif ic, sensitive, stable and high-throughput assay (Saccaggi et al. 2008; Zhang et al. 2012) and has proven effective for many species, including mealybugs. For example, Saccaggi et al. (2008) developed a multiplex PCR to successfully distinguish Planococcus citri (Risso), Planococcus f icus (Signoret) and Pseudococcus longispinus (Targioni Tozzetti). Hosseini and Hajizadeh (2011) designed a species-specif ic primer pair based on COI to identify the pests P. citri, Pseudococcus viburni (Signoret) and Pseudococcus comstocki (Kuwana). Tian et al. (2013) reported a PCR-based method to identify and monitor Phenacoccus solenopsis (Tinsley) by using SS-COI primers.

In our study, a P. manihoti-specif ic primer pair was designed based on mtDNA COI sequences from P. manihoti and related species. The specif icity and sensitivity of the primer sets were tested, and a SS-PCR assay for reliable and effective identif ication was validated by using P. manihoti from different geographic areas and host plants.

2. Materials and methods

2.1. Mealybug collection

A total of 22 mealybug species were included (Table 1). Adults and immature stages of P. manihoti were from Vietnam (donated by the Plant Protection Department Service (PPDS), Vietnam), Thailand (donated by the Department of Agricultural Extension (DOAE), Thailand) and Laos (donated by the Plant Protection Center, Ministry of Agriculture and Forestry, Laos), all major cassava exporting countries. Specimens of P. manihoti were identif ied by DNA barcoding (Wang 2016) and verif ied by morphological characteristics as described by Parsa et al. (2012). The 21 other mealybug species were obtained from f ield collection or were intercepted at ports of entry to China (Table 1) and identif ied prior to molecular studies using taxonomic keys (Tang 1992) and DNA barcoding (Wang 2016). All samples were stored in 99.7% ethanol at -20°C until DNA extraction. Voucher specimens were deposited in the Center for Management of Invasive Alien Species, Ministry of Agriculture and Rural Affairs of China.

2.2. DNA extraction

DNA was extracted from female adults of each of the 22 mealybug species (including 40 individuals of P. manihoti and six individuals of each of the 21 other species) (Table 1), as well as from eggs and nymphs (including 1st, 2nd, and 3rd instars) of P. manihoti (10 individuals of each developmental stage) following the protocols developed by Shi et al. (2005) with minor modif ication. Before DNA extraction, mealybugs (excluding eggs and 1st instar nymphs) were individually soaked in trichloromethane for 30 min, and then in ultrapure water for 6-10 h and dried on a piece of sterile f ilter paper.

The pretreated mealybugs were individually homogenized on a piece of paraf ilm (Bemis, Neenah, WI, USA) using a sterile plastic micropestle in 20 μL of ice-cold DNA extraction buffer (100 mmol L-1NaCl, 10 mmol L-1Tris-HCl, 50 mmol L-1EDTA, 0.5% SDS, and 0.2% β-mercaptoethanol, p H 8.0). The homogenate was placed in a 1.5-mL Eppendorf tube. The homogenizer (paraf ilm and plastic micropestle) was washed twice with 90 μL of ice-cold DNA extraction buffer, and the washed homogenate was put in the same Eppendorf tube. A total of 5 μL of proteinase K (20 mg mL-1) was added into the tube, and the homogenate was vortexed brief ly and incubated in a water bath at 65°C for 40 min, with a remixing after 20 min. After incubation, the tubes were boiled for 5 min to inactive the proteinase K. The lysate was centrifuged for 10 min at 7 500×g. The supernatant was transferred to a new 1.5-mL Eppendorf tube. After adding 150 μL of ice-cold NaCl (5 mol L-1) and agitating brief ly, the solution was centrifuged for 15 min at 12 000×g at 4°C. The supernatant was transferred to a new Eppendorf tube and mixed with one volume (200 μL) of ice-cold isopropanol and incubated for 30 min at -20°C. After centrifugation at 12 000×g at 4°C for 15 min, the DNA pellet was washed with ice-cold 75% ethanol and centrifuged for 10 min at 10 000×g at 4°C, dried before it was re-suspended in 40 μL ultrapure water (20 μL for eggs; 30 μL for 1st, 2nd or 3rd instars), and stored at -20°C. The concentrations of extracted DNA from a single individual P. manihoti adult female were assayed by spectrophotometry (NanoPhotometerTMP330, Implen, Munich, Germany).

Table 1 Details of the mealybug species used in the present study

2.3. Design of P. manihoti-specif ic COl primers

One pair of P. manihoti-specif ic primers, namely PMSSZW-1F and PMSSZW-1R, was designed using software Oligo 7 based on COI sequences from GenBank Database or the Database of Invasive Alien Species in China (DIASC) of P. manihoti (accessions KY611346-KY611349 (present study), KP692622, and KP6926223) and of 29 other mealybug species including 11 congeneric species (Appendix A). The sequence of the upstream primer (PMSSZW-1F) was 5′-CTTGATAAAACAGGAATTGAG-3′, and the sequence of the downstream primer (PMSSZW-1R) was 5′-CCTTTGATGATTTCTTCTTCT-3′. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The length of the amplif ication product was 353 bp.

2.4. PCR conditions

PCR amplif ication was set up in a total volume of 25 μL, including 1.0 μL of DNA, 2.5 μL of 10× reaction buffer, 0.4 μL of d NTPs (10 mmol L-1), 0.3 μL of Taq DNA polymerase (2.5 U μL-1), 0.4 μL of each primer (10 μmol L-1), and 20.0 μL of ultrapure water. PCR consisted of 38 cycles of 30 s at 94°C, 30 s at 51°C and 30 s at 72°C, after an initial denaturation for 2 min at 95°C. The 38 cycles were followed by a 5 min f inal extension at 72°C. PCR products (4 μL) were resolved in 1.0% agarose gel at 100 V for 40 min. To conf irm that the amplif ied products generated by the specif ic primers were from the target COI gene, each positive product was sequenced bi-directionally (Beijing Sunbiotech Co., Ltd., China).

2.5. Tests of primer specif icity and stability

To test the DNA quality, one pair of mealybug universal primers, PCoF/Lep R1 (COI), was used to amplify mtDNA (Park et al. 2011) from all mealybug specimens following the protocols developed by Park et al. (2011). Specif icity of the primer pair PMSSZW-1F and PMSSZW-1R was tested by performing PCR assays on 21 non-target mealybug species that are often intercepted at port of entry to China (Table 1), using P. manihoti DNA as the positive control. Negative controls containing all the reagents but with DNA replaced by ultrapure water were routinely processed. To determine the reliability of the primer set, PCR assays were performed on six individuals per mealybug species. In total, 132 individuals were analyzed. Moreover, to test the stability of the SS-COI primer pair, PCR assays were performed randomly in three models of thermal cycler: VeritiTM96-well Thermal Cycler (Applied Biosystems Inc., CA, USA), Gene Amp?PCR System 9700 (Applied Biosystems Inc., CA, USA) and Bio-Rad T100TMThermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.6. Tests of specif ic primer effcacy and sensitivity

To estimate the eff icacy of the primer set, DNA from a single female adult from each of three geographic populations and two host plant populations (Table 1) as well as individual immatures (including egg, 1st, 2nd and 3rd instars) of P. manihoti was assayed. To evaluate the lowest amount (i.e., detection threshold) of P. manihoti DNA detectable with this PCR assay, a DNA dilution series was used. The DNA concentrations were prepared at (69.2±7.5)×103, 34.6×103, 17.3×103, 8.7×103, 4.3×103, 2.2×103, 1.1×103, 540.8, 270.4, 135.2, 67.6, and 33.8 pg μL-1dilution, equal to 1/40, 1/80, 1/160, 1/320, 1/640, 1/1 280, 1/2 560, 1/5 120, 1/10 240, 1/20 480, 1/40 960, and 1/81 920 dilution of one female adult. PCR assays were performed on 10 individuals per population from different geographic areas and host plant species, developmental stage, and dilution series. In total, 100 individuals were analyzed separately. Moreover, two PCR products of P. manihoti from each population were sequenced.

3. Results

3.1. Verif ication of DNA quality

When DNA was amplif ied using the mealybug universal primer pair PCoF/Lep R1 (Park et al. 2011), a ～710 bp fragment was obtained, indicating that the extracted DNA was of high quality. The PCR amplicons of P. manihoti and 21 other mealybug species using the COI universal primer pair (PCoF and LepR1) are shown in Fig. 1. Therefore, any lack of reaction in subsequent tests did not result from a lack of mtDNA.

3.2. SS-COl primer specif icity and stability

The specif icity of the SS-COI primer pair PMSSZW-1F and PMSSZW-1R was assessed, resulting in a single 353-bp fragment obtained only from the DNA of P. manihoti, with no cross-reaction detected for the 21 other included mealybug species (Table 1). The mtDNA amplif ication pattern of P. manihoti and the 21 other mealybug species using the SSCOI primer pair is shown in Fig. 2. Moreover, the stability of this primer set was tested by using three different models of PCR thermal cycler, with only the 353-bp fragment detected from the DNA of P. manihoti adult. The amplif ication pattern of the mtDNA of P. manihoti adults using the SS-COI primer pair amplif ied on three different models of PCR thermal cycler is shown in Fig. 3.

Fig. 1 PCR amplicons of DNA from Phenacoccus manihoti and 21 other mealybug species using the COI universal primer pair. M, DNA marker; 1, P. manihoti, numbered as No. 1-1 in Table 1; 2-22, the other mealybug species, numbered as No. 2 to 22 in Table 1; 23, negative control.

3.3. COl sequence analysis

To conf irm the specif ied region of the P. manihoti amplicon, PCR products representing cassava mealybug from three different geographic populations (Thailand, Vietnam, and Laos) and two host plant populations (M. esculenta and Cucurbita moschata Duch.) were sequenced. Sequence analysis of the PCR products indicated that the P. manihoti amplicon was 353 bp (Fig. 4). All the 353-bp target fragment sequences were 100% identical. The signif icant alignments produced between this sequence and mitochondrial sequence data conf irmed that this amplicon is representative of the COI region. Furthermore, a BLAST search revealed signif icant identity (99-100%) between the 353-bp target sequence and published sequences for P. manihoti, while ＜96.3% identity was observed between P. manihoti and the other species, including 11 congeners.

3.4. Sensitivity of SS-COl primers

When the SS-COI primers PMSSZW-1F and PMSSZW-1R were used to test individual immatures (including eggs and 1st, 2nd, and 3rd instars) of P. manihoti, the 353-bp target fragment of P. manihoti was detected in all replicates. The amplif ication pattern of the SS-COI diagnostic marker for mtDNA of P. manihoti from different developmental stages is shown from lanes 1 to 5 in Fig. 5. Furthermore, the single marker fragment was successfully amplif ied in populations from two different host plant species, M. esculenta and C. moschata, and three different countries, including Vietnam, Thailand, and Laos (Fig. 5, lanes 6 to 9).

3.5. Detection effcacy of SS-COl primers

The detection eff icacy of the P. manihoti-specif ic primer pair was tested with a serial dilution of genomic DNA ranging from 69.2×103to 33.8 pg μL-1. The detection limit was determined at a dilution of 135.2 pg μL-1(equal to 1/20 480 of a single female adult) of P. manihoti DNA for all replicates. Detection of P. manihoti DNA at 67.6 pg μL-1was found successfully in eight of the 10 extracts. Positive responses for all samples were detected with 69.2×103, 34.6×103, 17.3×103, 8.7×103, 4.3×103, 2.2×103, 1.1×103, 540.8, 270.4, and 135.2 pg μL-1of P. manihoti DNA. The detection eff icacy for P. manihoti DNA using the SS-COI primer pair is shown in Fig. 6.

4. Discussion

Phenacoccus manihoti is a small, cryptic insect with strong negative impacts on cassava crops and a high capability for long-distance dispersal, easily spread via international trade (Parsa et al. 2012). A rapid and accurate identif ication method is required to prevent further spread and damage. The PCR assay developed here for P. manihoti identif ication by using the SS-COI primer set of PMSSZW-1F and PMSSZW-1R is a species-specif ic, rapid, sensitive, stable, and reliable assay. This PCR assay can be completed successfully within 2.5 h using unknown DNA from any developmental stage, and the presence of the 353-bp single fragment is suff icient to demonstrate detection of P. manihoti. Furthermore, this PCR assay requires no time-consuming slide preparation or further sequencing or restriction digestion. This enables non-specialists to accurately recognize P. manihoti among related mealybug species during plant quarantine, even when adult females are absent.

Fig. 2 Amplif ication pattern of mtDNA of Phenacoccus manihoti and 21 other mealybug species using the SS-COI primer pair. M, DNA marker; 1, P. manihoti, numbered as No. 1-1 in Table 1; 2-22, the other mealybug species, numbered as No. 2 to 22 in Table 1; 23, negative control.

Fig. 3 Amplif ication pattern of mtDNA from Phenacoccus manihoti adults using the SS-COI primer pair on three different equipments. M, DNA marker; 1-3, Veriti TM 96-well Thermal Cycler; 5-7, Gene Amp? PCR System 9700; 9-11, Bio-Rad T100TM Thermal Cycler; 4, 8 and 12, negative control.

Fig. 5 Amplif ication pattern of the SS-COI diagnostic marker for mtDNA of Phenacoccus manihoti from different developmental stages and populations. M, marker; 1-5, samples feeding on Manihot esculenta from Vietnam; 1, egg; 2, 1st instar nymph; 3, 2nd instar nymph; 4, 3rd instar nymph; 5, female adult; 6-9, samples from different countries and host plant species, numbered as No. 1-1 and No. 1-4 in Table 1; 10, negative control.

Fig. 6 Detection eff icacy for Phenacoccus manihoti DNA using the SS-COI primer pair. M, DNA marker; 1-12, 69.2×103, 34.6×103, 17.3×103, 8.7×103, 4.3×103, 2.2×103, 1.1×103, 540.8, 270.4, 135.2, 67.6, and 33.8 pg μL-1, equal to 1/40, 1/80, 1/160, 1/320, 1/640, 1/1 280, 1/2 560, 1/5 120, 1/10 240, 1/20 480, 1/40 960, and 1/81 920 of a whole female adult of P. manihoti, respectively; 13, negative control.

We tested the specif icity of the SS-COI primer set with 21 closely related mealybug species frequently intercepted at ports of entry to China or common in the f ield, including four quarantine pests (P. solenopsis, Dysmicoccus neobrevipes Beardsley, Planococcus lilacinus (Cockerell), and Planococcus minor (Maskell)) and three congeners (Phenacoccus madeirensis Green, Phenacoccus solani Ferris, and P. solenopsis). No cross reaction was detected across non-target species, validating the specif icity of the primer pair (Fig. 2). Moreover, this approach was effective for identif ication of all developmental stages and across different populations of P. manihoti (Fig. 5) and can be performed with different models of PCR thermal cycler (Fig. 3), demonstrating the robust stability of the SS-COI primer pair. Successful amplif ication was obtained with the detection limit of 135.2 pg μL-1of DNA (Fig. 6), indicating high sensitivity in identif ication of P. manihoti.

Our f indings align with those from Saccaggi et al. (2008), Park et al. (2010), Hosseini and Hajizadeh (2011), and Tian et al. (2013). Though RAPD markers can distinguish P. manihoti from closely related species, Phenacoccus herreni Cox and Williams or P. madeirensis (Calatayud and Le Rü 2006), RAPD analysis is weak in repeatability and reliability (Black 1993), restricting wide use. High sensitivity and stability is the most important superiority of our species-specif ic COI PCR assay over the RAPD PCR assay.

This PCR system could facilitate testing of imported plants and plant materials for the presence of P. manihoti, with ovisacs, eggs, newly hatched nymphs, and females that can be present in international cargo (Malumphy 2005; Wu and Wang 2011; Parsa et al. 2012) and often show a high degree of morphological similarity to related species (Wu and Wang 2011; Parsa et al. 2012). In addition, this simple procedure does not require extensive taxonomic or molecular experience. To ensure applicability, the specif icity of the primer pair (PMSSZW-1F and PMSSZW-1R) should be further tested with a wider range of congeneric mealybug species.

5. Conclusion

Phenacoccus manihoti is a signif icant invasive pest which

has not yet invaded China, and thus research efforts must focus on detection and prevention. We developed SS-COI markers that allow for quick and reliable identif ication of all ontogenetic stages. This represents a considerable improvement over morphological identif ication methods and will facilitate customs inspection and monitoring, prevention, and management of this potential invasive pest. Furthermore, this assay will be benef icial in understanding the mechanisms of worldwide dispersal and management of this pernicious mealybug species.

We are grateful to Ms. Guo Rong from the National Agro-Tech Extension and Service Center, for providing specimens of P. manihoti from Vietnam and Thailand; Prof. Hou Maolin from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), for providing specimens of P. manihoti from Laos; Prof. Zhou Pei from the Wuxi Entry-Exit Inspection and Quarantine Bureau, China for providing specimens of Planococcus minor from Singapore and Prof. Ma Jun from the Guangdong Entry-Exit Inspection and Quarantine Bureau, China for providing the specimens Ferrisia gilli from the USA. This work was supported by the National Key R&D Program of China (2017YFC1200600, 2016YFC1201200 and 2015BAD08A16) and the Science and Technology Innovation Program of CAAS (caascx-2013-2018-IAS).

Appendixassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm